Mixture Of Bovine Serum Albumin Research Articles

Selective enrichment of glycopeptides based on copper tetra(N-carbonylacrylic) aminephthalocyanine and iminodiacetic acid functionalized polymer monolith.

Efficient separation and enrichment of low-abundance glycopeptides from complex biological samples is the key to the discovery of disease biomarkers. In this work, a new material was prepared by coating copper tetra(N-carbonylacrylic) aminephthalocyanine and iminodiacetic acid onto poly(glycidyl methacrylate-pentaerythritol triacrylate) monolith. The monolith was applied to polymer monolithic microextraction for specific capture of glycopeptides coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The developed monolith exhibited satisfactory efficiency for glycopeptide enrichment with high selectivity and detection sensitivity. When the tryptic digest of immunoglobulin G was used as the sample, total 24 glycopeptides were identified and the detection limit was determined as 5fmol. When the approach was applied to the analysis of glycopeptides in the mixture of bovine serum albumin and immunoglobulin G (100:1, m/m) digests, 16 glycopeptides could still be observed. Moreover, the monolith was successfully applied to the selective enrichment of glycopeptides from human serum digests, exhibiting great practicability in identifying low-abundance glycopeptides in complex biological samples.

Preparation of novel phenyl boronic acid functionalized silica gel using triazo-cyanide click chemistry and its application in glycoprotein/glycopeptide selective enrichment

The application of boronic acid affinity chromatography to glycoprotein/glycopeptide enrichment is increasingly maturing. The enrichment selectivity, biocompatibility, and facile operation protocol are key aspects in efficient enrichment methods. In this work, a novel triazo-cyanide boronic acid functionalized material (TCNBA) was prepared using triazo-cyanide click chemistry. The TCNBA was proved to be successfully synthesized through infrared ray (IR) characterization. Subsequently, the glycopeptide/glycoprotein enrichment selectivity of the TCNBA was evaluated. Matrix-assisted laser desorption/ionization time-of-flight mass (MALDI-TOF MS) was employed for the glycopeptide enrichment selectivity evaluation. Taking the digestion of horseradish peroxidase (HRP) and immunoglobulin G (IgG) as samples, 13 and 11 glycopeptides could be characterized with improved signals after TCNBA enrichment, respectively. High abundance non-glycopeptides could be removed effectively from the eluting fraction. This result indicates the high glycopeptide enrichment selectivity of TCNBA. In addition, a mixture of HRP and bovine serum albumin (BSA) enzymatic solution (1:10, amount of substance ratio) was utilized as a sample, and five glycopeptide signals could be identified following enrichment. To evaluate the glycoprotein enrichment selectivity, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE) was adopted as an evaluation method. Mixtures of HRP, IgG, BSA, and ribonuclease B (RNaseB) proteins were employed as samples, and the results demonstrated that TCNBA had a high glycoprotein enrichment selectivity. The application of TCNBA to the analysis of a real biosample was also evaluated using human plasma. The results indicated the TCNBA could be utilized in large-scale glycoprotein analysis.

Quantitative Analysis of Membrane Fouling Mechanisms Involved in Microfiltration of Humic Acid–Protein Mixtures at Different Solution Conditions

A systematical quantitative understanding of different mechanisms, though of fundamental importance for better fouling control, is still unavailable for the microfiltration (MF) of humic acid (HA) and protein mixtures. Based on extended Derjaguin–Landau–Verwey–Overbeek (xDLVO) theory, the major fouling mechanisms, i.e., Lifshitz–van der Waals (LW), electrostatic (EL), and acid–base (AB) interactions, were for the first time quantitatively analyzed for model HA–bovine serum albumin (BSA) mixtures at different solution conditions. Results indicated that the pH, ionic strength, and calcium ion concentration of the solution significantly affected the physicochemical properties and the interaction energy between the polyethersulfone (PES) membrane and HA–BSA mixtures. The free energy of cohesion of the HA–BSA mixtures was minimum at pH = 3.0, ionic strength = 100 mM, and c(Ca2+) = 1.0 mM. The AB interaction energy was a key contributor to the total interaction energy when the separation distance between the membrane surface and HA–BSA mixtures was less than 3 nm, while the influence of EL interaction energy was of less importance to the total interaction energy. The attractive interaction energies of membrane–foulant and foulant–foulant increased at low pH, high ionic strength, and calcium ion concentration, thus aggravating membrane fouling, which was supported by the fouling experimental results. The obtained findings would provide valuable insights for the quantitative understanding of membrane fouling mechanisms of mixed organics during MF.

Preparation of copper tetra(N-carbonylacrylic) aminephthalocyanine functionalized zwitterionic-polymer monolith for highly specific capture of glycopeptides.

In this work, poly(glycidyl methacrylate-ethyleneglycol dimethacrylate) monolith functionalized with copper tetra(N-carbonylacrylic) aminephthalocyanine and iminodiacetic acid was successfully synthesized. Owing to hydrogen bonding and hydrophilic interactions, the monolith exhibited good performance for glycopeptide enrichment. When the tryptic digests of horseradish peroxidase were enriched by the developed monolith, a total of 20 glycopeptides could be captured and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis with a detection limit as low as 0.5fmolμL-1. With the mixture of bovine serum albumin and horseradish peroxidase digests (200:1, m/m) as the sample, 14 glycopeptides were identified after enrichment, showing the high selectivity of the monolith. Moreover, the functionalized monolith exhibited good stability and reproducibility. It was successfully applied to enrich glycopeptides from human serum, demonstrating its potential applications in selective and efficient capture of glycopeptides in complex biological samples. Graphical abstract ᅟ.

Modeling water partition in composite gels of BSA with gelatin following high pressure treatment

Changes in the structural properties of hydrogels made with gelatin and bovine serum albumin mixtures were recorded following exposure to high pressure at 300 MPa for 15 min at 10 and 80 °C. Dynamic oscillation, SEM, FTIR and blending law modelling were utilised to rationalise results. Pressurization at the low temperature end yielded continuous gelatin networks supporting discontinuous BSA inclusions, whereas an inverted dispersion was formed at the high temperature end with the continuous BSA network suspending the discontinuous gelatin inclusions. Lewis and Nielsen equations followed the mechanical properties of the composites thus arguing that solvent partition between the two phases was always in favour of the polymer forming the continuous network. As far as we are aware, this is the first attempt to elucidate the solvent partition in pressurised hydrogel composites using blending law theory. Outcomes were contrasted with earlier work where binary mixtures were subjected only to thermal treatment.

Forward osmosis membrane performance during simulated wastewater reclamation: Fouling mechanisms and fouling layer properties

A thin-film composite (TFC) FO membrane was used to study the fouling characteristics of effluent organic matter (EfOM) fractions in treated wastewater effluent represented by alginate, bovine serum albumin, humic acid and octanoic acid. These model foulants were chosen to represent carbohydrates, proteins, humic substances and fatty acids. Inter foulant interactions and their influence on membrane flux loss was established by running series of fouling tests with feed solutions containing mixtures of model foulants. The obtained results demonstrated that under our experimental conditions humic acid (HA) and octanoic acid (OA) had no significant role on permeate flux loss during wastewater treatment over short periods. However, alginate and bovine serum albumin (BSA) and their mixtures caused significant total flux loss, through alginate-calcium complexation that led to the formation of a resistant gel layer on the membrane surface. Protein fouling was mainly attributed to multiple layer adsorption onto the polyamide membrane surface. Mixing alginate with BSA saw a further decline in permeate flux, worse than that caused individually by either of the foulants, and gives an indication of the synergistic effect between the two foulants. There were favourable inter-foulant interactions between the carbohydrates and the proteins that promoted the formation of hybrid aggregates that were deposited on the membrane surface and enhanced flux loss. The additional presence of humic acid to the mixture of BSA and alginate further aggravated membrane fouling. Polysaccharides and proteins were found to be the most dominant foulants during wastewater reclamation. The extent of interactions between the organic foulants had an effect on the fouling layer structure and its role in permeate flux loss.

Liquid Chromatography High-Resolution Mass Spectrometry Identifies the Glycation Sites of Bovine Serum Albumin Induced by d-Ribose with Ultrasonic Treatment.

Ultrasonication is an emerging technology applied in food processing and biological experimental pretreatments. Cavitation phenomena induced during ultrasonic treatment can generate localized high temperature and pressure, which can result in glycation reaction between protein and reducing sugars. In this study, the mixture of bovine serum albumin (BSA) and d-ribose was treated under 600 W for different times. Interestingly, a large amount of carbonized black materials appeared after ultrasonication, while the UV absorbance and intrinsic fluorescence spectra reflecting conformational changes were not obvious. Only 12 sites (11 lysines and 1 arginine) of the BSA with ribose under ultrasonic treatment for 35 min were identified through liquid chromatography high-resolution mass spectrometry (LCHR-MS). K547, K548, R359/R360, and K587 were the most reactive glycated sites, with the average degree of substitution per peptide molecule (DSP) value ranging from 15 to 35%. The glycated modification was distributed not only in domain III, but also in domains I and II. The glycated modification could occur during ultrasonic treatment, thereby influencing the properties of biomacromolecule after extraction.

Combined Layer/Particle Approaches in Surface Molecular Imprinting of Proteins: Signal Enhancement and Competition.

Herein we report novel approaches to the molecular imprinting of proteins utilizing templates sizing around 10 nm and some 100 nm. The first step comprised synthesizing nanoparticles of molecularly imprinted polymers (MIP) towards bovine serum albumin (BSA) and characterizing them according to size and binding capacity. In a second step, they were utilized as templates. Quartz crystal microbalances (QCM) coated with MIP thin films based on BSA MIP nanoparticles lead to a two-fold increase in sensor responses, compared with the case of directly using the protein as the template. This also established that individual BSA molecules exhibit different “epitopes” for molecular imprinting on their outer surfaces. In light of this knowledge, a possible MIP-based biomimetic assay format was tested by exposing QCM coated with BSA MIP thin films to mixtures of BSA and imprinted and non-imprinted polymer (NIP) nanoparticles. At high protein concentrations (1000 ppm) measurements revealed aggregation behavior, i.e., BSA binding MIP NP onto the MIP surface. This increased sensor responses by more than 30% during proof of concept measurements. At lower a BSA concentration (500 ppm), thin films and particles revealed competitive behavior.

Synthesis and chromatographic characteristics of iminodisuccinic acid-functionalized silica stationary phase

In this study, we developed a multifunctional chromatographic medium by using iminodisuccinic acid (IDS) as ligand, γ-glycidoxypropyltrimethoxysilane (γ-GLDP) as spacer arm and silica as matrix. The medium was characterized by fourier transform infrared spectrometry (FT-IR). Binding capacity of IDS on chromatographic medium was determined by potentiometric titration. The effect of mass ratios (w/w) between silica and IDS on the medium synthesis was discussed. The optimal ratio of matrix, spacer arm and ligand was 1.5g: 2.0mL: 4.6g. Based on different chromatographic modes, protein mixtures of bovine serum albumin, ribonuclease and lysozyme were successfully separated with IDS-Silica column and IDS-Cu(II)-Silica column, respectively. To examine metal chelating behavior of IDS-Silica stationary phase, optimal geometries and related parameters of complexes formed by IDS with Fe3+, Cu2+, Ni2+, Zn2+, Co2+ and Ca2+ were achieved by quantum computing. The stabilities of the complexes were predicted according to the results of theoretical calculation. Sorption capacities of IDS-Silica stationary phase for metal ions were determined by using offline frontal chromatography (FC) combined with inductively coupled plasma atomic emission spectrometry (ICP-AES). A good agreement was found between simulating results and experimental observations. The order of chelating strength of IDS-Silica medium for metal ions was Fe3+>Cu2+>Ni2+>Zn2+>Co2+>Ca2+. Compared with IDA-Silica, Asp-Silica and Glu-Silica stationary phases, IDS-Silica displayed a stronger chelating property for metal ion.

Facile and easily popularized synthesis of L-cysteine-functionalized magnetic nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides.

Protein glycosylation is one of the most important post-translational modifications. Also, efficient enrichment and separation of glycopeptides from complex samples are crucial for the thorough analysis of glycosylation. Developing novel hydrophilic materials with facile and easily popularized synthesis is an urgent need in large-scale glycoproteomics research. Herein, for the first time,a one-step functionalization strategy based on metal-organic coordination was proposed and Fe3O4 nanoparticles were directly functionalized with zwitterionic hydrophilic L-cysteine(L-Cys), greatly simplifying the synthetic procedure. The easily synthesized Fe3O4/L-Cys possessed excellent hydrophilicity and brief composition, contributing to affinity for glycopeptides and reduction in nonspecific interaction. Thus, Fe3O4/L-Cys nanoparticles showed outstanding sensitivity (25amol/μL), high selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a mass ratio of 100:1), good reusability (five repeated times), and stability (room temperature storage of 1month). Encouragingly, in the glycosylation analysis of human serum, a total of 376 glycopeptides with 393 N-glycosylation sites corresponding to 118 glycoproteins were identified after enrichment with Fe3O4/L-Cys, which was superior to ever reported L-Cys modified magnetic materials. Furthermore, applying the one-step functionalization strategy, cysteamine and glutathione respectively direct-functionalized Fe3O4 nanoparticles were successfully synthesized and also achieved efficient glycopeptide enrichment in human serum. The results indicated that we have presented an efficient and easily popularized strategy in glycoproteomics as well as in the synthesis of novel materials. Graphical abstract Fe3O4/L-Cys nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides.

New Ti-IMAC magnetic polymeric nanoparticles for phosphopeptide enrichment from complex real samples

The work describes the preparation of a new magnetic phase for batch enrichment of phosphopeptides. The material exploits the advantages of magnetic solid phase extraction and couples them with the most employed approach for phosphopeptide enrichment, i.e. Ti4+-IMAC. In order to immobilize Ti4+ ions on the surface of the magnetite nanoparticles, they were first covered by a silica shell and then modified to expose at the surface bromine containing groups. Glycidyl methacrylate was subsequently polymerized from these groups using the “grafting from” approach by the activator regenerated by electron transfer–atom transfer radical polymerization (ARGET-ATRP) technique. Finally, the glycidyl groups were reacted with iminodiacetic acid to functionalize the material with moieties suitable for coordination. The prepared material was extensively characterized and subsequently tested for enrichment of a bovine serum albumin mixture with casein to ascertain its potential. With positive results, the new magnetic polymeric material was further employed to set up an enrichment method on yeast protein digest based on shotgun proteomics. The sample to phase ratio was optimized and the best condition compared to a commercial TiO2 spin column. At the end of the comparison, the new material proved better and could enrich a larger total number of phosphopeptides with increased selectivity. All these conclusions and the test performed on a real complex sample within the final shotgun application further support the applicability of the new material in phosphopeptide analysis of real matrices.

Changes in structure and performance during diafiltration of binary protein solutions due to repeated cycles of fouling/alkaline cleaning

The purpose of this work was to evaluate the effect of the temperature (50 and 60°C) of a NaOH cleaning solution during the diafiltration of a binary mixture of bovine serum albumin and β-lactoglobulin, through a 300kDa tubular ceramic membrane along repeated operational cycles. To this aim, final permeate volume, membrane and fouling resistances and individual protein concentration were analyzed. At the end of each individual study, the membranes were characterized by liquid–liquid displacement porosimetry. As a result, 50°C was found to be the most appropriated temperature due to its higher capability to restore the initial membrane resistance and the higher efficiency achieved in terms of protein separation. Both conditions fulfilled without altering the structural properties of the membrane as given by porosimetric analysis. In contrast, a great fouling resistance involving null protein transmission occurred when cleaning was performed at 60°C.

Highly selective enrichment of phosphorylated proteins by using Spore@Fe3+ microspheres

The advantages of spore-based microspheres include high monodispersity, presence of different functional groups (carboxylic, amino, and hydroxyl groups), facile industrial-scale preparation by using cell cultures via fermentation in a potentially cost effective and environment friendly manner, and high uniformity. We developed a novel spore@Fe3+ microsphere for specific capture of phosphoproteins. Caseins (α-casein and β-casein) as phosphoproteins were used to evaluate binding capacity and enrichment factor. The spore@Fe3+ microspheres demonstrate high binding capacity and selectivity for phosphoproteins (1983 and 1818 mg g−1 for α-casein and β-casein, respectively). A mixture of bovine serum albumin and β-casein at 100:1 ratio displayed an enrichment factor higher than 173-fold, which can nearly be considered “purification” of phosphoproteins. The proposed method is a promising technique in developing more selective, rapid, low cost, and high-throughput platforms for phosphoprotein enrichment, and it presents potential application in investigation of protein functions and in personalized diagnostic tests.

Biomimetic synthesis of calcium carbonate with different morphologies and polymorphs in the presence of bovine serum albumin and soluble starch

Calcium carbonate has been synthesized by the reaction of Na2CO3 and CaCl2 in the presence of bovine serum albumin (BSA) and soluble starch. Effects of various bovine serum albumin (BSA) and soluble starch on the polymorph and morphology of CaCO3 crystals were investigated. Crystallization of vaterite is favored in the presence of BSA and soluble starch, respectively, while calcite is favored in the presence of a mixture of BSA and soluble starch. The morphologies of CaCO3 particles in the presence of mixture of BSA and soluble starch are mainly rod-like, suggesting that the BSA, soluble and their assemblies play key roles in stabilizing and directing the CaCO3 crystal growth.

Effect of surface energy of solid surfaces on the micro- and macroscopic properties of adsorbed BSA and lysozyme

The surface energy, a macroscopic property, depends on the chemical functionality and micro- and macroscopic roughness of the surface. The adsorption of two widely used proteins bovine serum albumin (BSA) and lysozyme on surfaces of four different chemical functionalities were done to find out the interrelation between macroscopic and microscopic properties. We have observed the secondary structure of protein after its adsorption. In addition, we observed the variation of surface energy of proteins due to variation in adsorption time, change in protein concentration and effect of a mixture of proteins.Surfaces of three different chemical functionalities namely, amine, hydroxyl and octyl were obtained through self-assembled monolayer on silica surfaces and were tested for responses towards adsorption of lysozyme and BSA. The adsorbed lysozyme has higher surface energy than the adsorbed BSA on amine and octyl surfaces. On hydroxyl functional surface, the surface energy due to the adsorbed lysozyme or BSA increases slowly with time.The surface energy of the adsorbed protein increases gradually with increasing protein concentration on hydrophobic surfaces. On hydrophilic surfaces, with increasing BSA concentration in bulk solution, the surface energy of the adsorbed protein on GPTMS and amine surfaces is maximum at 1μM concentration. During the adsorption from a mixture of BSA and lysozyme on octyl surface, first lysozyme adsorbs and subsequent BSA adsorption leads to a high surface energy.

Selective enrichment of glycopeptides/phosphopeptides using Fe3O4@Au-B(OH)2@mTiO2 core-shell microspheres

In this work, the bifunctional Fe3O4@Au-B(OH)2@mTiO2 core-shell core-shell microspheres were designed and synthesized for the selective enrichment of glycopeptides/ phosphopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the microspheres were successfully applied to the enrichment of phosphopeptides and glycopeptides, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine β-casein diluted to 2.0pgμL−1 (8.0×10–17molμL−1) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 80pgμL−1 (2.0×10–15molμL−1). The enrichment selectivity of the bifunctional microspheres was evaluated by capturing phosphopeptides from a peptide mixture of β-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (4.2×10–12mol of β-casein and 4.2×10−9mol of BSA in 100μL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:100 (5.0×10–12mol of HRP and 5.0×10−10mol of BSA in 100μL).

Characterization of adsorption and lubrication of synovial fluid proteins and HA on DLC joint bearings surface

Adsorption and competitive adsorption behavior of bovine serum albumin (BSA), human γ-globulin (HGG), and hyaluronic acid (HA) molecules on a diamond-like carbon (DLC) film surface were investigated by the means of a quartz crystal microbalance with dissipation (QCM-D), and antigen-antibody reactions. The results showed that a BSA monolayer could be formed on the DLC film surface with side-on orientation and that an HGG monolayer could be formed on the DLC film surface with end-on orientation by using pure BSA or HGG solutions, respectively. HA molecules were hardly adsorbed onto the surface of DLC film or HGG or BSA biomolecular layer. The results of competitive adsorption showed that HGG molecules could displace BSA molecules and that HGG was the dominantly adsorbed molecule on the DLC film surface in a mixture of BSA, HGG, and HA. The frictional behavior of a DLC film sliding against Al2O3 in a mixture of BSA, HGG, and HA, was investigated by means of the friction coefficient and an open circuit potential, which suggested that the HGG molecule was the main component that formed the lubricating film.

The development of a weak anion micro-capillary film for protein chromatography

In this study, the surface of a microporous walled micro-capillary film (MMCF) was modified into a weak anion exchanger by coupling cyanuric chloride and 2-diethylaminoethylamine (DEAE) to the ethylene-vinyl alcohol (EVOH) matrix. Fourier transform infrared spectroscopy (FTIR) measurements of modified and unmodified MMCFs confirmed the addition of a triazine ring and DEAE onto the membrane. Binding of bovine serum albumin (BSA) at pH 7.2 was found to follow a Langmuir isotherm with a maximum equilibrium binding of 12.4mg BSA/mL adsorbent and 8.2mg BSA/mL adsorbent under static and flow conditions, respectively. The ion exchange capacity, determined by Mohr’s titration of chlorine atoms displaced from the functionalised surface, was found to be 195±21μmol Cl−/mL of adsorber, comparable to commercial ion exchangers. BSA adsorption onto the ion exchanger was strongly pH-dependant, with an observed reduction in binding above pH 8.2.Frontal experiments of a BSA (5mg/mL) and lysozyme (5mg/mL) mixture demonstrated successful separation of BSA from lysozyme at more than 97% purity as verified by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Separation between similarly charged anionic molecules was also achieved using BSA (5mg/mL) and herring sperm DNA (0.25mg/mL). BSA was extracted at 100% purity, demonstrating the ability of MMCF-DEAE to remove significant DNA contamination from a protein solution. These experiments highlight the potential for MMCFs to be used for fast protein purification in preparative chromatography application.

Model to predict inhomogeneous protein–sugar distribution in powders prepared by spray drying

A protein can be stabilized by spray drying an aqueous solution of the protein and a sugar, thereby incorporating the protein into a glassy sugar matrix. For optimal stability, the protein should be homogeneously distributed inside the sugar matrix. The aim of this study was to develop a model that can predict the distribution of protein and sugar in an evaporating droplet using bovine serum albumin (BSA) and trehalose as model protein and sugar, respectively. This was achieved by expanding a previously developed model that was able to predict the growth or shrinkage of inhaled droplets in the airways (Grasmeijer, Frijlink & Hinrichs, 2016). The droplet was considered to consist of a finite number of concentric spherical shells in which the change in concentration of components was calculated in time, enabling the prediction of concentration gradients inside the droplet. It was found that during evaporation of the droplet, an inhomogeneous protein–sugar distribution was formed even when surface active properties were not considered. The relatively large protein molecule was predicted to accumulate much faster at the surface of the droplet than the sugar due to slower diffusion, resulting in a lower sugar/protein ratio at the surface of the particle than in the center. For a mixture of BSA and trehalose, not considering surface active properties, it was predicted that 60% of protein was incorporated in the powder at a lower sugar/protein ratio than when protein and sugar would have been homogeneously distributed, which may hamper efficient protein stabilization. These predictions can be used to more accurately adapt the initial composition of the solution to ensure proper stabilization of the protein, for example by simply increasing the amount of sugar to increase the sugar/protein ratio at the surface. Or, if this is limited by the desired loading, changing the drying conditions to slow down the drying rate.

Synthesis of bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets for sequential selective enrichment of phosphopeptides and glycopeptides for mass spectrometric analysis.

In this work, the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets were designed and synthesized for the sequential selective enrichment of phosphopeptides and glycopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the nanosheets were successfully applied to the enrichment of phosphopeptides and glycopeptides sequentially, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine β-casein diluted to 0.02ng/μL (8 × 10(-16) mol/μL) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 0.1ng/μL (2.5 × 10(-15) mol/μL). The enrichment selectivity of the bifunctional nanosheets was evaluated by capturing phosphopeptides from a peptide mixture of β-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (8.3 × 10(-12) mol of β-casein and 8.3 × 10(-9) mol of BSA in 100μL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:50 (5.0 × 10(-11) mol of HRP and 2.5 × 10(-9) mol of BSA in 100μL). Graphical Abstract A workflow of the sequential enrichment strategy for phosphopeptides and glycopeptides by the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets.